ABSTRACT Fiber-cell membranes of the human eye lens are saturated with cholesterol (showing cholesterol-to- phospholipid mole ratios from 1 in the cortex to as high as 4 in the nucleus), which leads to the formation of immiscible cholesterol crystalline domains (CCDs) within these membranes. At the highest cholesterol content, up to 50% of the cell-membrane surface can be occupied by CCDs. These conditions exist in the lens nucleus, where the cholesterol content can significantly exceed the solubility threshold of the membrane. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens can CCDs play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. The long-term objective of this proposal is to achieve a greater understanding of cholesterol's function in fiber-cell membranes. In the short-term, we will (i) examine how high cholesterol content affects the lateral organization of phospholipid lens membranes, with special attention paid to the formation of CCDs, and (ii) test the hypothesis that the presence of CCDs determines the properties of the surrounding bulk phospholipid- cholesterol membranes. (iii) Additionally, experiments will reveal if the phospholipid composition of the lens membrane affects the formation, size, and stability of CCDs. (iv) Finally, the methodology developed and tested in model-membrane systems will be applied to detect and characterize coexisting cholesterol crystalline and bulk phospholipid-cholesterol domains in lens fiber-cell membranes during maturation, aging, and cataract formation. For studies of coexisting domains in membranes isolated from the eye lens, as well as in models of fiber-cell membranes, the discrimination by oxygen transport (DOT) method will be used. The DOT method, which is based on electron paramagnetic resonance (EPR) spin-labeling techniques, permits discrimination of different membrane domains and gives information about structure and molecular dynamics as a function of the membrane depth in coexisting domains without the need for their separation. It also allows information about oxygen transport within and across membrane domains to be obtained.